Association of genetic variations in FoxP3 gene with Graves' disease in a Southwest Chinese Han population

Abstract Background Graves' disease (GD) is a T cell‐mediated organ‐specific autoimmune disease. Forkhead box P3 (FoxP3) is an excellent marker for the induction and development of regulatory T cells (Tregs). Recent studies showed that single‐nucleotide polymorphisms (SNPs) in the FoxP3 gene were associated with the increased susceptibility to several autoimmune diseases. In the present study, we investigated the association of FoxP3 gene polymorphisms with GD in a Southwest Chinese Han population. Methods A two‐stage case‐control study was performed in 890 healthy controls (male, 282; female, 608) and 503 patients with GD (male, 138; female, 365). Four SNPs (rs3761548, rs3761549, rs3761547, and rs2280883) were genotyped by the polymerase chain reaction‐restriction fragment length polymorphism assay. The χ 2 test was used to compare the genotype distributions and allele frequencies between GD patients and healthy controls. Results In the first stage, the significantly increased frequencies of the A allele (p = .031, odds ratio [OR] = 1.635) and AA genotype (p = .023, OR = 3.257), together with a significantly decreased frequency of the C allele (p = .031, OR = 0.611) of FoxP3/rs3761548 were found in female patients with GD. None of the other FoxP3 SNPs was associated with GD susceptibility. Subsequent validation and combination of data confirmed the association between FoxP3/rs3761548 and the female patients with GD (A allele: p < .001, OR = 1.672; AA genotype: p = .005, OR = 2.488; CC genotype: p = .001, OR = 0.622; C allele: p < .001, OR = 0.615, respectively). Conclusion Our findings suggest that FoxP3/rs3761548 is significantly associated with female GD patients in a Southwest Chinese Han population.


| INTRODUCTION
Graves' disease (GD), also known as toxic hyperthyroidism, is the most common cause of hyperthyroidism. It is an autoimmune disease caused by thyrotropin receptor antibody produced by the thyroidstimulating hormone receptor. GD is also a multisystem syndrome charactered by hypermetabolic syndrome, diffuse goiter, eye sign, skin lesions, and thyroid acromegaly. GD is the most common in patients between the ages of 20 and 40 year, with a male: female prevalence of 1:4−1:6. 1 The annual incidence rate is approximately 20−30/1,000,000. 2 Although the pathogenesis of GD has not been fully elucidated, it is believed to be the result of the interaction between genetic susceptibility and environmental factors. 3,4 Therefore, the identification of genes and sites contributing to GD susceptibility is an important issue in the study of the pathogenesis of this disease.
Regulatory T cells (Tregs) play a pivotal role not only in the suppression of immune responses and the development of immune tolerance but also in balancing immune responses by suppressing immune hyperreactivity through the secretion of inhibitory cytokines or direct cell contact-dependent mechanisms. 5 Th17/Treg homeostasis is disrupted in many autoimmune diseases. 6 Previous studies showed functional defects in Tregs and increased numbers of Th17 cells in GD patients, 7,8 while another study showed that Treg levels were reduced in GD patients compared with healthy controls, suggesting that Tregs play an important role in the pathogenesis of GD. 9 Forkhead box P3 (FoxP3) is a transcription factor specifically and stably expressed in Tregs. The FoxP3 gene is located on human chromosome Xp11.23. Polymorphisms in FoxP3 may change its expression level and weaken the inhibitory function of Tregs, thus affecting the role of Tregs in the immune response and potential autoimmune diseases. 10 The rs3761547, rs3761548, rs3761549, and rs2280883 single-nucleotide polymorphisms (SNPs) are related to FoxP3 expression. 11 The role of FoxP3 polymorphisms in the development of GD is not well understood. Independent studies in China, Poland, India, and Japan have shown that certain FoxP3 mutations are associated with GD susceptibility. [12][13][14][15][16] However, another study found there was no association between FoxP3 polymorphism and GD susceptibility in United Kingdom population. 17 Although previous studies showed that FoxP3 polymorphism plays a critical role in the pathogenesis of GD, the results are inconsistent due to differences in environmental conditions or genetic backgrounds. Study by Zheng et al. 13 on FoxP3 gene polymorphisms were associated with GD susceptibility among Han population in central plains of China. However, to date, there has been no comprehensive investigation into the association between FoxP3 polymorphisms and GD susceptibility in the Southwest Chinese Han population. Therefore, we explored whether the FoxP3 SNPs (rs3761548, rs3761549, rs3761547, and rs2280883) were associated with GD susceptibility in a Southwest Chinese Han population.

| Study subjects
A total of 503 GD patients and 890 healthy controls were recruited from the Affiliated Hospital of Zunyi Medical University and the Yongchuan Hospital of Chongqing Medical University. The cases and controls were recruited from January 2016 to December 2020. All patients were diagnosed according to the 2016 Guidelines of the American Thyroid Association and the Society of Clinical Endocrinologists for the Diagnosis and Treatment of Hyperthyroidism. 18 The controls were selected from the health examination centers of these two hospitals, and they did not have any other autoimmune disease and were matched for sex and age with the patients with GD. This study was also registered in the Chinese Clinical Trial Registry (registration number: ChiCTR1900022398). The written informed consent was obtained from all participants in the study before blood collection.

| DNA extraction
Venous blood samples were collected from patients with GD and healthy controls into EDTA anticoagulant tubes. Genomic DNA was extracted from peripheral blood using the QIAamp DNA Blood Mini Kit (Qiagen). The purity of DNA was determined according to the optical density ratio of 260/280 nm.

| Genotyping
The polymerase chain reaction-restriction fragment length polymorphism assay was conducted to genotype the four SNPs (rs3761547, rs3761548, rs3761549, and rs2280883) of FoxP3 gene, which were established to be associated with autoimmune diseases in previous studies. The target sequence was amplified by PCR with suitable primers. 13 The target sequence of each gene was amplified by PCR and the product was digested with specific restriction enzymes. Table 1 summarizes the sequences of the forward and reverse primers, PCR conditions and restriction enzymes. Five samples were randomly repeated for genotyping, and the results were the same as before.

| Statistical analysis
The statistical analyses were performed with the SPSS Statistics version 17.0 (SPSS, Inc.). χ 2 test was used to assess significant differences in genotype and allele frequencies among the groups. The association of SNPs with GD risk was evaluated by odds ratios (ORs) with 95% confidence intervals (CIs) for five different genetic models. Statistical significance was established at p < .05. For controls, SNPStats (http://bioinfo.iconcologia.net/ SNPStats/start. HTM) was used to analyze each SNP of Hardy-Weinberg equilibrium (HWE).

| Clinical features of the enrolled GD patients
The detailed demographic characteristics and clinical manifestations of the enrolled patients are shown in Supporting Information: There was no significant difference in age and sex between the control and GD groups (p > .05). In the control group, the genotype frequency distribution of the four studied SNPs (rs3761547, rs3761548, rs3761549, and rs2280883) did not deviate from the HWE.
3.2 | Allele and genotype frequencies of the four SNPs in the first phase of the study Considering that FoxP3 gene is located on the X chromosome, we divided the patients into two groups, namely, female and male. A two-stage case-control study was conducted. In the first stage study, we tested the association between the four SNPs (rs3761547, rs3761548, rs3761549, and rs2280883) of FoxP3 gene and GD susceptibility in 195 GD cases and 367 healthy controls. The results showed that significantly increased frequencies of A allele (p = .031, OR = 1.635) and AA genotype (p = .023, OR = 3.257) of FoxP3/rs3761548 were observed in female GD patients as compared to the healthy controls (Table 2). Moreover, as compared to the healthy male group, GD male patients had a lower frequency of FoxP3/rs3761548 A allele (p = .049, OR = 0.453) and had a higher frequency of FoxP3/rs3761548 C allele (p = .049, OR = 2.209) ( Table 3). None of the other SNPs, including rs3761547, rs3761549 and rs2280883, were associated with GD susceptibility (p＞.05; Supporting Information: Table S2, Table S3).
T A B L E 1 Primers, reaction conditions and restriction enzymes used for PCR-RFLP analysis.

| DISCUSSION
This study investigated the association between FoxP3 gene polymorphism and GD in a Southwest Chinese Han population. It was found that there is a significant association between FoxP3/rs3761548 and female GD patients, while the rs3761547, rs3761549 and rs2280883 were not correlated to GD susceptibility. Based on the comparison between the female control group and the female GD group, we observed significant differences in the frequencies of FoxP3/rs3761548 A allele and AA genotype. Among these four candidate polymorphisms (rs3761548, rs3761547, rs3761549, and rs2280883), only the association of rs3761548 with GD susceptibility in female GD patients was identified in the first stage of our T A B L E 2 Allele and genotype frequencies of FoxP3/rs3761548 in female GD patients and healthy controls.  [20][21][22][23][24][25][26][27][28] A recent metaanalysis of susceptibility to various autoimmune diseases showed an association between rs3761548 and autoimmune disease. 29 An additional recent meta-analysis showed that the rs3761548 polymorphism of the FoxP3 was associated with increased GD risk in Asians, due to the Tregs suppression and the enhancement of autoimmune responses. 30 Similarly, our recent meta-analysis also showed that rs3761548 was associated with GD in Asians in the subgroup analysis according to ethnicity. 31 In the second stage of our study, we found that there were significant differences in the frequencies of the FoxP3/rs3761548 A allele, AA genotype, AC genotype, CC genotype and C allele between female healthy controls and GD patients. The FoxP3/rs3761548 polymorphisms the AA genotype was also reported to be a risk factor for several diseases, including MS, acute coronary syndrome, systemic lupus erythematosus, vitiligo, allergy and thyroid cancer. 27,[32][33][34][35][36] It is found that the FoxP3/rs3761548 AA genotype was correlated with a significant decrease in the FoxP3 protein level in the Tregs of generalized vitiligo patients. 22 Additionally, FoxP3/rs3761548 A allele has been identified to be significantly associated with BD, UC, MS, and RA. 21,23,37,38 It was demonstrated that the A allele of FoxP3/rs3761548 affected Tregs function, which is one of the factors involved in the susceptibility for MS in females. 26 Earlier research has established a link between the FoxP3/rs3761548 and an elevated risk of severe osteoarthritis in the Turkish population. 39 More recently, a study discovered that the C allele of the rs3761548 increased the susceptibility to HT. 28 However, intriguingly, no significant difference was observed in the prevalence of rs3761548 between GD and control groups in both Indian and British populations. 16,17 Other research also indicated that there was no notable difference in the incidence of FoxP3/rs3761548 among healthy and GD groups in children and adolescents. 14 These disparities in results could be attributed to the varying populations studied. Our study proposes that there may be differences in the frequency of FoxP3/ rs3761548 among GD patients from diverse populations, suggesting it could be a risk factor for Asian GD patients. Regarding the other three FoxP3 variations (rs3761549, rs3761547, and rs2280883), our data revealed no significant differences in allele and genotype frequencies between case and control groups, which was consistent with other previous studies. [12][13][14][15][16]40 However, previous research by Yu et al. 12 and Zheng et al. 13 demonstrated an association between the rs2280883 variant and GD susceptibility in both the Zhejiang Han and Hubei Han populations. A possible explanation for these inconsistent results is that the sample sizes in these studies were small and the research subjects were distributed in different regions of China.

Gene
Moreover, our combined study observed significant differences between the female control group and the female GD group in the frequencies of the FoxP3/ rs3761548 A allele and AA genotype. In contrast, no significant correlation between FoxP3/rs3761548 and GD was found in the male population in the combined study.
T A B L E 3 Allele frequencies of FoxP3/rs3761548 in male patients with GD and male healthy controls. A deeper understanding of these findings can be achieved by examining the location of the FoxP3 gene on the X chromosome and its correlation with GD. Various potential mechanisms, including probabilistic and X-inactivation, could account for the higher incidence of GD in women at the X chromosomal locus. 41 As for the application value of our finding in clinical work, we believe that understanding the genetic basis of GD might help identify individuals at risk, which could guide early intervention and treatment decisions. The specific role of FoxP3/rs3761548 polymorphism in this regard requires further investigation. Several limitations of our study need to be specified. First, we investigated only a limited number of SNPs in FoxP3 gene. It is possible that there are other SNPs in FoxP3 gene to be associated with GD susceptibility. Second, only Southwest Chinese Han population was enrolled, and it is not certain whether our findings can be generalized to other ethnic populations. Third, there are limited numbers of subjects in each subgroup according to gender and study stage, which might reduce the test power of this study. Furthermore, there are lack of functional study on the mechanism of FoxP3/rs3761548 polymorphism in regulating Tregs and Th17/Treg homeostasis.

| CONCLUSION
In summary, our results suggested that FoxP3/rs3761548, but not the rs3761547, rs3761549 and rs2280883, is associated with an increased risk of GD in female patients in a Southwest Chinese Han population. Further studies are needed to elucidate the role of FoxP3/ rs3761548 and Th17/Treg homeostasis in the development of GD.